Search Results (1,440)

In this work, an innovative Schönflies motion generator manipulator is introduced, featuring a parallel architecture composed of serial chains with mixed degrees of freedom. Fundamental kinematic aspects essential to any manipulator such as displacement, velocity, acceleration, and singularity analyses are thoroughly addressed. Screw theory is employed to derive compact input–output expressions for velocity and acceleration, leveraging the properties of reciprocal screws and lines associated with the constrained degrees of freedom in the parallel manipulator. A key advantage of the proposed design is its near-complete avoidance of singular configurations, which significantly enhances its applicability in robotic manipulation. Numerical examples are provided to validate the theoretical results, with corroboration from specialized tools such as ADAMS™ software and data fitting algorithms. These results confirm the reliability and robustness of the developed kinematic analysis approach. Full article

Background/objective: Mucosal melanoma (MM) is a poorly responsive, rare and aggressive subtype with few cases having targetable recurrent driver mutations, although Ras/MAPK and PI3K/AKT/mTOR signaling pathway activations are common. Eventual tumor evasion of targeted therapy continues to limit treatment success. Adequate models are necessary to address therapeutic resistance. The relatively greater incidence of naturally occurring MM in dogs, as well as its comparable clinical and pathological characteristics to human MM, represents an opportunity for study as a human MM patient surrogate. Resistance-promoting crosstalk between Ras/MAPK and PI3K/AKT/mTOR signaling under trametinib inhibition of MEK was studied in canine MM. Emphasis was placed on the suppressive effect of trametinib on cell cycle entry and its potential role in drug resistance. Methods: D-type cyclins were investigated following trametinib treatment of five MM cell lines exhibiting differential drug sensitivities. Signaling pathway activation, proliferation, survival, cell death, and cell cycle were analyzed in the context of D-type cyclin expression. Cyclin D2 expression was manipulated using siRNA knockdown or inducible recombinant overexpression. Results: Trametinib diminished cyclin D1 in all cell lines. While relatively trametinib-resistant MM cells exhibited capacity to upregulate cyclin D2, which promoted proliferation, sensitive MM cells lacked similar cyclin D2 compensation. Inhibition of the compensatory cyclin D2 in resistant cells conferred sensitivity. Induced cyclin D2 overexpression in otherwise trametinib-sensitive MM cells promoted survival. Upregulated PI3K/AKT/mTOR signaling under trametinib treatment was suppressed by mTORC1/2 inhibition, which similarly diminished cyclin D2 response. Conclusions: The compensatory switch from preferential reliance on cyclin D1 to D2 plays a role in MM resistance to MEK inhibition. Full article

Plant triterpenoids are structurally diverse specialized metabolites with significant ecological, medicinal, and agricultural importance. Oxidosqualene cyclases (OSCs) catalyze the crucial cyclization step in triterpenoid biosynthesis, generating the fundamental carbon skeletons that determine their structural diversity and biological functions. Genome-wide identification of OSC genes was performed using bioinformatics tools, including HMMER and BLASTP, followed by phylogenetic analysis, gene structure analysis, conserved domain and motifs identification, cis-regulatory element prediction, protein–protein interaction analysis, and expression profiling using publicly available transcriptome data from UV-B treated A. annua six-week-old seedlings. We identified 24 AaOSC genes, classified into CAS, LAS, LUS, and unknown subfamilies. Phylogenetic analysis revealed evolutionary relationships with OSCs from other plant species. Gene structure analysis showed variations in exon–intron organization. Promoter analysis identified cis-regulatory elements related to light responsiveness, plant growth and development, hormone signaling, and stress response. Expression profiling revealed differential expression patterns of AaOSC genes under UV-B irradiation. This genome-wide characterization provides insights into the evolution and functional diversification of the OSC gene family in A. annua. The identified AaOSC genes and their regulatory elements lay the foundation for future studies aimed at manipulating triterpenoid biosynthesis for medicinal and biotechnological applications, particularly focusing on enhancing stress tolerance and artemisinin production. Full article

Crocin biosynthesis involves a complex network of enzymes with biosynthetic and modifier enzymes, and the manipulation of these pathways holds promise for improving human health through the broad exploitation of these bioactive metabolites. Crocins play a significant role in human nutrition and health, as they exhibit antioxidant and anti-inflammatory activity. Plants that naturally accumulate high levels of crocins are scarce, and the production of crocins is highly limited by the characteristics of the crops and their yield. The CCD2 enzyme, initially identified in saffron, is responsible for converting zeaxanthin into crocetin, which is further modified to crocins by aldehyde dehydrogenases and glucosyltransferase enzymes. Crops like tomato fruits, which naturally contain high levels of carotenoids, offer valuable genetic resources for expanding synthetic biology tools. In an effort to explore CCD2 enzymes with improved activity, two CCD2 alleles from saffron and Crocosmia were introduced into tomato, together with a UGT gene. Furthermore, in order to increase the zeaxanthin pool in the fruit, an RNA interference construct was introduced to limit the conversion of zeaxanthin to violaxanthin. The expression of saffron CCD2, CsCCDD2L, led to the creation of transgenic tomatoes with significantly high crocins levels, reaching concentrations of 4.7 mg/g dry weight. The Crocosmia allele, CroCCD2, also resulted in high crocins levels, reaching a concentration of 2.1 mg/g dry weight. These findings underscore the importance of enzyme variants in synthetic biology, as they enable the development of crops rich in beneficial apocarotenoids. Full article

The BCR-ABL1 chimeric oncoprotein plays a pivotal role in the pathogenesis of Chronic Myeloid Leukemia (CML) as its constitutive kinase activity transforms the hematopoietic stem cell, promoting pro-survival signaling. We and others have previously shown that the manipulation of BCR-ABL1 catalytic activity modulates its intracellular localization, thereby transforming the culprit of CML into a pro-apoptotic protein that selectively kills leukemic cells. Here, we investigated the role of the BCR coiled-coil and C2 domains on BCR-ABL1 intracellular localization and leukemogenic potential. We performed a bioinformatic analysis that identified two putative nuclear localization signals (NLSs) in BCR. Using recombinant DNA strategies, we generated multiple BCR and BCR-ABL1 mutants that were ectopically expressed in human cells. The intracellular localization of each construct was analyzed by immunofluorescence, while their biological activity was investigated employing proliferation and transforming assays. We show that BCR displays two nuclear localization signals functionally inactivated by the coiled-coil and C2 domains. The removal of these regions reactivated the nuclear migration of both BCR and BCR-ABL1 mutants. Moreover, BCR-ABL1 constructs devoid of the coiled-coil and C2 domains displayed reduced transforming potential in Ba/F3 cells and in primary human CD34+ progenitors. Finally, we demonstrate that the deletion of the C2 domain compromises TKI efficacy. Our findings identify two nuclear localization signals in the BCR sequence that are functionally suppressed by the coiled-coil and C2 domains. Targeting these regions may provide additional therapeutic strategies to manipulate both BCR-ABL1 intracellular localization and kinase activity. Full article

Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP^KO, CANX^KO, and CALR^KO cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies. Full article

The rapid growth of the Internet of Things (IoT) has revolutionized various industries by enabling interconnected devices to exchange data seamlessly. However, IoT systems face significant security challenges due to decentralized architectures, resource-constrained devices, and dynamic network environments. These challenges include denial-of-service (DoS) attacks, anomalous network behaviors, and data manipulation, which threaten the security and reliability of IoT ecosystems. New methods based on machine learning have been reported in the literature, addressing topics such as intrusion detection and prevention. This paper proposes an advanced anomaly detection framework for IoT networks expressed in several phases. In the first phase, data preprocessing is conducted using techniques like the Median-KS Test to remove noise, handle missing values, and balance datasets, ensuring a clean and structured input for subsequent phases. The second phase focuses on optimal feature selection using a Genetic Algorithm enhanced with eagle-inspired search strategies. This approach identifies the most significant features, reduces dimensionality, and enhances computational efficiency without sacrificing accuracy. In the final phase, an ensemble classifier combines the strengths of the Decision Tree, Random Forest, and XGBoost algorithms to achieve the accurate and robust detection of anomalous behaviors. This multi-step methodology ensures adaptability and scalability in handling diverse IoT scenarios. The evaluation results demonstrate the superiority of the proposed framework over existing methods. It achieves a 12.5% improvement in accuracy (98%), a 14% increase in detection rate (95%), a 9.3% reduction in false positive rate (10%), and a 10.8% decrease in false negative rate (5%). These results underscore the framework’s effectiveness, reliability, and scalability for securing real-world IoT networks against evolving cyber threats. Full article

The large portion of the eukaryotic genomes was considered non-functional and called the “dark matter” of the genome, now appearing as regulatory hubs coding for RNAs without the potential for making proteins, known as non-coding RNA. Long non-coding RNA (lncRNA) is defined as functional RNA molecules having lengths larger than 200 nucleotides without the potential for coding for proteins. Thousands of lncRNAs are identified in different plants and animals. LncRNAs are characterized by a low abundance, fewer exons than mRNA, tissue-specific expression, and low sequence conservation compared to protein-coding genes (PCGs). LncRNAs, like PCGs, are regulated by promoters and enhancers with characteristic chromatin signatures, DNA methylation, multiple exons, introns, and alternate splicing. LncRNAs interact with DNA, mRNA, microRNA, and proteins, including chromatin/histone modifiers, transcription factors/repressors, epigenetic regulators, spliceosomal, and RNA-binding proteins. Recent observations indicate that lncRNAs code for small peptides, also called micropeptides (<100 amino acids), and are involved in the development and growth of plants, suggesting the bi-functional activities of lncRNAs. LncRNAs have emerged as the major regulators of diverse functions, principally by altering the transcription of target genes. LncRNAs are involved in plant growth, development, immune responses, and various physiological processes. Abiotic, biotic, nutrient, and other environmental stresses alter the expressions of numerous lncRNAs. Understanding the mechanisms of actions of lncRNAs opens up the possibility of improving agronomic traits by manipulating lncRNAs. However, further studies are required in order to find the interactions among the deregulated lncRNAs and validate the findings from high-throughput studies to harness their potential in crop improvement. Full article

Human corneal endothelial cell therapy has recently emerged as a novel solution to treat corneal endothelial diseases. We previously demonstrated the potential of utilizing non-cultured primary corneal endothelial cells (CEnCs) isolated from donor corneas with low endothelial cell density for simple non-cultured endothelial cell injection (SNEC-I) therapy. This study aimed to develop a robust and semi-automated approach for cell counting, characterize the extent of cellular manipulation, and evaluate the translational workflow. To address this, we evaluated manual and automated cell counting approaches and characterized the extent of manipulation of CEnCs through the analysis of cell cycle status, gene expressions, and transcriptomic profiles with single-cell RNA-sequencing. The translational feasibility and functionality of SNEC-I therapy were examined using an established rabbit model of bullous keratopathy. Manual hemocytometry and automated cell-counters exhibited comparable accuracy and reproducibility. Analysis of cell cycle status, cell cycle genes (n = 11), and transcriptomic profiles revealed close resemblance between the native corneal endothelium and its donor-matched SNEC-I-harvested cells. Successful resolution of bullous keratoplasty in the pre-clinical model supports the feasibility, efficacy, and safety of SNEC-I therapy. In conclusion, SNEC-I therapy serves as an attractive corneal endothelial therapeutic approach (from a regulatory standpoint) in view of the minimal extent of cellular manipulation. Full article

Trichogramma wasps, egg parasitoids widely used to control lepidopteran pests, have long eluded in-depth molecular mechanistic studies due to their minute size and genetic tool scarcity. While previous RNAi efforts were restricted to T. dendrolimi, we developed the first cross-species RNAi system for both T. dendrolimi and the previously intractable T. ostriniae. Temporal expression profiling identified white and laccase 2 as stage-specific RNAi targets, peaking during prepupal/pupal stages, which were tested across species and developmental stages using microinjection and soaking dsRNA delivery methods. Survival analysis prioritized soaking for T. dendrolimi prepupae/pupae, while microinjection was essential for T. ostriniae to bypass prepupal mortality during soaking. Concentration-dependent RNAi targeting the white gene achieved 85.61% transcript reduction in T. dendrolimi via soaking and 89.36% in T. ostriniae via microinjection at 2000 ng/μL, correlating with 64.06% and 32.09% white-eyed pupae, causing a significant reduction in eye pigments. For the laccase 2 gene, soaking at 2000 ng/μL induced 88.35% transcript reduction in T. dendrolimi and 73.31% in T. ostriniae, leading to incomplete cuticle tanning and sclerotization. This study resolves the long-standing challenge of genetic manipulation in Trichogramma wasps, providing a universally applicable framework to decipher parasitoid–host interactions at the molecular scale, which is useful for sustainable pest management strategies. Full article

Yam (Dioscorea spp.) provides various nutritional and medicinal benefits, including a high starch content, dietary fiber, essential micronutrients, and bioactive compounds. The molecular mechanisms underlying tuber expansion have not yet been clarified. Rapid alkalinization factor (RALF) genes, which mediate various processes in plants, are thought to contribute to the regulation of tuber growth; however, their role in yam development, especially in gibberellin (GA)-mediated processes, remains unclear. Here, we characterized seven DrRALF genes in the yam genome. Analysis of gene duplication demonstrated that the expansion of DrRALF genes was primarily driven by whole-genome duplication or segmental duplication. Phylogenetic analysis revealed that DrRALF genes were concentrated in specific clusters, indicating that their functions are relatively conserved. DrRALF5 was specifically expressed in the roots, and DrRALF2, DrRALF3, DrRALF4, and DrRALF6 were highly expressed in flowers. DrRALF1, DrRALF2, DrRALF3, DrRALF4, DrRALF5, and DrRALF6 were shown to play a role in tuber expansion. Subsequent qRT-PCR validation of four selected DrRALF genes confirmed the regulation of DrRALF2, DrRALF4, DrRALF5, and DrRALF6 by GA and PP333 (paclobutrazol, a GA biosynthesis inhibitor). Yeast one-hybrid assays further showed that the DrRALF6 promoter region interacted with the GA-signaling protein, DrDELLA1. Our findings provide novel insights into the regulatory network controlling yam tuber expansion, especially through the interaction between DrRALF6 and GA signaling pathways. Our results clarify the molecular mechanisms involved in tuber growth and propose a promising strategy for improving yam production through genetic manipulation of the GA-RALF signaling pathway. Full article

Blue light (BL) plays a crucial role in regulating floral transition and can be precisely manipulated in controlled-environment agriculture (CEA). However, previous studies on BL-mediated flowering in CEA have produced conflicting results, likely due to species-specific responses and variations in experimental conditions (such as light spectrum and intensity) as summarized in our recent systematic review. This speculation still lacks a mechanistic explanation at the molecular level. By synthesizing recent advances in our understanding of the signaling mechanisms underlying floral transition, this review highlights how both internal signals (e.g., hormones, carbohydrates, and developmental stage) and external cues (e.g., light spectrum, temperature, nutrients, stress, and magnetic fields) are integrated into the flowering pathway mediated by BL. Key signal integration nodes have been identified, ranging from photoreceptors (e.g., cryptochromes) to downstream components such as transcription factors and central flowering regulator, FLOWERING LOCUS T (FT). This signal integration offers a potential mechanistic explanation for the previously inconsistent findings, which may arise from interspecies differences in photoreceptor composition and variation in the expression of downstream components influenced by hormonal crosstalk, environmental conditions, and developmental stage, depending on the specific context. This review provides novel molecular insights into how BL modulates floral transition through interactions with other signals. By systematically compiling and critically assessing recent research findings, we identify key research gaps and outline future directions, particularly the need for more studies in agriculturally important crops. Furthermore, this review proposes a conceptual framework for optimizing BL-based lighting strategies and exploring underexamined interaction factors in the regulation of flowering. Full article

The creation of transgenic chickens holds significant promise for the agricultural and biotechnological sectors, offering potential improvements in disease resistance and production efficiency. The preferred method for generating gene-edited chickens involves the genetic manipulation of primordial germ cells (PGCs), making the identification and isolation of these cells a growing focus of research. PGCs are the precursors to sperm and oocytes, responsible for transmitting genetic material to the next generation. In humans, PGCs are characterized by their large size, round nuclei, and refractive lipids in the cytoplasm, and can be identified using periodic acid–Schiff (PAS) staining and the surface marker stage-specific embryonic antigen 1 (SSEA1). Similarly, chicken PGCs express SSEA1, but their most specific marker is the chicken vasa homologue (CVH), the avian equivalent of the RNA-binding factor gene vasa. However, SSEA1, along with other known surface markers, does not bind to all PGCs or lacks specificity, while CVH, although highly specific to PGCs, is intracellular and unsuitable for isolating viable cells. This study aims to develop an antibody targeting a PGC surface marker with the same specificity as CVH. Despite the importance of identifying surface markers for PGC characterization, to date, such reagents are limited. To address this, whole chicken PGCs were injected into mice, leading to the generation of a panel of monoclonal antibodies. One antibody was found to bind cultured chicken PGCs and showed reduced expression upon differentiation with retinoic acid, indicating its specificity to PGCs. Immunoprecipitation followed by mass spectrometry identified the antigen as myosin heavy chain-like (MYH9) protein. The antibody, αMYH9, was further characterized and shown to bind circulating PGCs and embryonic gonadal PGCs (Hamburger Hamilton (H-H) stage 30, embryonic day 6.5–7). Whilst our primary aim was to determine the binding to PGCs, further investigation is required to determine potential binding to somatic cells. In conclusion, this study provides the characterization of a surface marker for chicken PGCs, with significant implications for advancements in avian genetic preservation, agriculture, and biotechnology. Full article

Phytopythium vexans is a plant pathogen responsible for a variety of destructive diseases in crops worldwide. This includes patch canker, damping-off, root, and crown rots in economically important crops, such as apple, pear, grapevine, citrus, avocado, and kiwi. The pathogen has a global distribution, and a recent report confirmed its presence in southern Ontario, Canada. This study presents the first genome sequencing, assembly, and annotation of the Canadian P. vexans strain SS21. To explore how variation in secreted protein repertoires may relate to infection strategies and host adaptation, we compared the predicted secretome of SS21 with reference strains from Iran (CBS 119.80) and China (HF1). The analysis revealed that HF1 harbors a larger set of CAZymes, sterol-binding proteins, and predicted effectors, which may suggest broader adaptive potential. In contrast, strain SS21 appears to have adapted to a niche-specific strategy, with fewer necrosis-inducing proteins, glucanase inhibitors, and effectors, possibly indicating adaptation to specific hosts or ecological conditions. Comparative genome data highlight distinct evolutionary trajectories that may have shaped each strain’s infection strategy, with SS21 potentially serving as a robust additional reference for future studies on P. vexans biology and host interactions. While this analysis identifies key candidate effectors, gene expression studies are required to validate their functional roles in infection and host manipulation. Full article

This review critically examines the evolving landscape of genetic animal models for investigating cardiovascular diseases (CVDs). We analyze established models, including spontaneously hypertensive rats, Watanabe hyperlipidemic rabbits, etc., and transgenic models that have advanced our understanding of essential and secondary hypertension, atherosclerosis, and non-ischemic diseases of the heart. This review systematically evaluates the translational strengths and physiological limitations of these approaches across species barriers. Particular attention is paid to emerging technologies—AAV-mediated gene delivery, CRISPR-Cas9 editing, and chemogenetic tools—that enable unprecedented precision in manipulating cardiac-specific gene expression to study pathophysiological mechanisms. We address persistent challenges including off-target effects and transgene expression variability, while highlighting innovations in synthetic vectors and tissue-specific targeting strategies. This synthesis underscores how evolving genetic technologies are revolutionizing cardiovascular research paradigms, offering refined disease models and optimized therapeutic interventions that pave the way toward personalized medicine approaches for the world’s leading cause of mortality. Full article